The p53 tumor suppressor is a homotetrameric, sequence-specific transcription factor that has crucial roles in apoptosis, cell cycle arrest, DNA repair, cellular senescence, metabolism and tumor suppression. It is maintained at low levels in unstressed cells, but becomes stabilized and activated following DNA damage through extensive post-translational modification (PTM). Our research has focused on identifying and exploring the biological roles of p53 PTMs to better understand how they modulate p53 functions. Previously, we characterized the complexes formed between the Taz2 domain of the transcriptional co-activator p300 and either the first (TAD1, residues 1-40) or second (TAD2, residues 35-59) transactivation subdomains of p53. Our results showed that both TAD1 and TAD2 occupy the same region of Taz2, form short alpha helices when bound, have similar affinities for Taz2, and are stabilized by both hydrophobic and electrostatic interactions. Although both TAD1 and TAD2 bind to various domains within p300, they also interact with distinct proteins and can function independently of one another. These results suggest the existence of distinguishing transcriptional cofactors for TAD1 and TAD2 whose interaction is regulated differently by p53 phosphorylation. Comparison of the structures of the two complexes also suggests that these two similar domains within p53 may function differently in co-activator recruitment after stress. Therefore, we have initiated a project to identify differential interactors of TAD1 and TAD2 in response to cellular stress that are modulated by p53 phosphorylation. We have synthesized a series of p53 peptides representing either TAD1 or TAD2 to use as bait for pulldown of interacting proteins from nuclear extracts prepared from cells treated with etoposide; reductive dimethylation of peptides followed by mass spectrometry analysis has been used to identify and quantitatively compare the interactors to discriminate between those that preferentially interact with TAD1 and TAD2. Our preliminary experiments using biological triplicate pulldowns have identified a list of potential interactors that show a preference for either unmodified or modified p53 in untreated cells or following etoposide treatment. In addition to several known binding partners of p53 TAD1, we have identified several that have not previously been investigated as p53 interactors. We are proceeding to validate the mass spectrometry findings and further characterize the biological implications of these interactions. The C-terminus of p53 exhibits a diverse array of post-translational modifications, including phosphorylation, methylation, acetylation, ubiquitination, sumoylation, neddylation and hydroxylation that are primarily localized to the terminal thirty residues of the protein. We are interested in understanding the specific effects of individual site-specific modifications and the interplay between them. We have reported that p53 can be both mono- and dimethylated on Lys382, with the former modification repressing p53 transcriptional activity and the latter promoting DNA repair, in addition to demonstrated acetylation and ubiquitination of the same site. Recently, using a high-content imaging siRNA screen and a chemical screen, we identified SETD8, a monomethylase for both lysine 20 on histone H4 and p53 lysine 382, as a suppressor of p53 activity in neuroblastoma cell lines. Genetic or pharmacological inhibition of SETD8 activity resulted in activation of the p53 wild-type pathway by decreasing p53K382me1. This work, performed in collaboration with Drs. Veschi and Thiele of the CCR, was recently published in Cancer Cell. In addition, we have synthesized long synthetic peptides containing specific PTMs on Lys382 that will be used in biochemical and biophysical assays to investigate the differential effects of these modifications on p53 conformation and interactions. p53 point mutations have been reported to occur in approximately half of all tumors, with marked over-representation of specific hot-spot residues. These mutations leave p53 unable to function as a transcription factor and prevent tumor growth. Moreover, many mutant forms of p53 have novel oncogenic activities due to a gain-of-function mechanism. Therefore, mutant p53 is an important target for the development of an agent to improve the response to anti-cancer treatments. One characteristic of many of the hot-spot p53 mutants is their structural instability with partial unfolding and the formation of aggregates similar to those seen in amyloid diseases, thus resulting in protein inactivation. The development of effective inhibitors of mutant p53 protein aggregation requires the ability to determine the state of aggregation of the p53 protein in cells. We are using a suite of detection tools to probe the conformational and aggregation status of p53 in cultured cells using the high-throughput, high-resolution Opera imaging system. Chemical libraries available through NCATS will be screened as concentration series and scored for their ability to reduce the formation of p53-specific aggregated protein oligomers. Our study will evaluate the potential of small molecules alone and in combination with nutlin-3a, an inhibitor of the interaction of p53 with its negative regulator MDM2, to specifically reactivate mutant p53 and induce cell cycle arrest and apoptosis through the wildtype p53 pathway.